The epidermis is the outermost layer of the skin. Its cells are differentiated to provide a waterproof mechanical barrier, crucial to enable life on Earth. Due to its contact with the outside, the epidermis suffers damage more frequently and directly than any other tissue in the body, therefore its organization depends on the mechanisms ability to repair and renew.
The epidermis has a stratified organization with three layers or principal strata (stratum basale, stratum granulosum and stratum corneum), and is due to the strong balance between the proliferation and differentiation of keratinocytes. When the cells in the basal layer undergo differentiation they lose their proliferative capacity and move progressively closer to the stratum corneum. The stratum basale is composed of keratinocytes, and their characteristic activity is the synthesis of keratin, a protein which forms intermediary filaments and is responsible for giving the epidermis its hardness. Several layers of spur cells are found on this stratum basale layer and a stratum granulosum cell layer is located on the spur cell layers. The granulosum cell layer is crucial for maintaining the impermeability of the epidermis, which is its most important function. The granulosum cell layer, furthermore, marks the limit between metabolically active cells and dead cells. Such dead cells result from the progressive loss of organelles and the filling of its cytoplasms with keratin as they advance towards the outside, the cells being reduced to flat scales completely filled with densely packed keratin. These dead cells become detached from the surface of the skin approximately one week after emerging from the basal layer. This particular organization of the stratum corneum serves to protect the skin, and in turn allows it to maintain a certain level of flexibility retaining a defined quantity of water. Hydration of the stratum corneum is crucial to determine the appearance, metabolism, mechanical properties and the skin's function as a barrier.
Aging of the skin is a complex process which comprises two differentiated processes, intrinsic and extrinsic aging. The former is due to genetic factors and does not just affect the skin but all the body's organs. Extrinsic aging is caused by environmental factors, such as exposure to contamination, tobacco smoke, ultraviolet radiation, wind, cold climates, etc. Both processes overlap on areas of the skin exposed to the outside, and share chemical processes. Aging and skin aging is understood to be the appearance of visible changes to the appearance of the skin, as well as those which are discernible by touch, such as and not restricted to, the development of discontinuities on the skin such as wrinkles, fine lines, expression lines, stretch marks, striae, furrows, irregularities or roughness, increase in the size of pores, loss of hydration, loss of elasticity, loss of firmness, loss of smoothness, loss of the capacity to recover from deformation, loss of resilience, sagging of the skin such as sagging cheeks, the appearance of bags under the eyes or the appearance of a double chin, among others, changes to the color of the skin such as marks, reddening, bags or the appearance of hyperpigmented areas such as age spots or freckles among others, anomalous differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange-peel skin, loss of collagen structure and other histological changes of the stratum corneum, of the dermis, epidermis, vascular system (for example the appearance of spider veins or telangiectasias) or of those tissues close to the skin.
One of the clearest signs of aging is the appearance of wrinkles, due both to the loss of collagen and the loss of the skin's elasticity. At an ultrastructural level, the skin's collagen network becomes denser with age, despite the loss of total collagen content. The elastic fibers progressively decline and break into fragments, which leads to the loss of the skin's elasticity and the appearance of wrinkles. The process begins in phases relatively early on in life, and accelerates after forty. It has been proven that women lose 2.1% of their collagen level per year after the menopause and that 30% of the collagen is lost in the first five years after the menopause [Brincat M. et al., “A study of the decrease of skin collagen content, skin thickness, and bone mass in the postmenopausal woman” Obstet. Gynecol., (1987), 70, 840-845]. Therefore, an increase in the quantity of wrinkles and fine lines is observed, a loss of flexibility of the skin and women experience a feeling of “dry skin” or tight skin.
This process is aggravated by the action of the family of matrix metalloproteinases (MMP), a family of proteolytic enzymes (endoproteinases) which can collectively degrade the macromolecular components of the extracellular matrix (collagen and elastin) and the basal lamina. The degradation of the collagen fibers leads to skin with a sagging and wrinkly appearance, especially in the areas exposed to sunlight, such as the skin on the face, ears, neckline, scalp, hands and arms, which is not desirable.
Furthermore, prolonged exposure to ultraviolet radiation, particularly UVA and UVB, stimulates MMP synthesis, which destroy collagen [Fisher G. J. et al., “Pathophysiology of premature skin aging induced by ultraviolet light”. New Eng. J. Med., (1997), 337, 1419-1429; Fisher G. J., “Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo”. J. Invest. Dermatol., (2001), 117, 219-226], which is one of the principal causes of photoaging.
One of the causes of different conditions, disorders and diseases of the skin and/or mucous membranes are found in low levels of collagen, either due to its diminished synthesis, or due to the increase in its degradation. Among them we can highlight chronic ulcers, psoriasis [Flisiak I. et al., “Effect of psoriasis activity on metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in plasma and lesional scales”. Acta Derm Venereol., (2006), 86, 17-21], oral conditions such as gingivitis and periodontitis, skin cancer [Kerkelä E. et al., “Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer”. Exp Dermatol., (2003), 12, 109-125], metastasis [Sato H. et al., “Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis”. Cancer Sci., (2005), 96, 212-217], dermatitis [Katoh N. et al., “Increased levels of serum tissue inhibitor of metalloproteinase-1 but not metalloproteinase-3 in atopic dermatitis”. Clin. Exp. Immunol., (2002), 127, 283-288], rosacea, telangiectasia, couperosis, bags under the eyes, periorbital dark circles, varicose veins, alopecia [Jarrousse F. et al., “Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth”. Int. J. Dermatol., (2001), 40, 385-392] cellulitis, orange peel skin, healing or re-epithelialization disorders, and stretch marks, among others. Dermatitis includes skin conditions, disorders or diseases which cause inflammation, such as contact dermatitis, atopic dermatitis, sensitive skin and eczema. Therefore, all these conditions, disorders and diseases are treatable with compounds stimulating collagen synthesis.
The loss of hydration in aged and photoaged skin is another of the causes of the appearance of wrinkles on the skin, as well as the change to the skin barrier function. The skin's water content can influence lipid synthesis [Rawlings A. V. et al., “Abnormalities in stratum corneum structure lipid composition and desmosome degradation in soap-induced winter xerosis”. J. Soc. Cosmet. Chem., (1994), 45, 203-220], in epidermal DNA synthesis [Denda M. et al., “Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses”. J. Invest. Dermatol., (1998), 111, 873-878], in its function as a barrier [Denda M. et al., “Exposure to a dry environment enhances epidermal permeability barrier function”. J. Invest. Dermatol., (1998), 111, 858-863] and in the thickness of the skin [Sato J. et al., “Dry condition affects desquamation of stratum corneum in vivo”. J. Dermatol. Sci., (1998), 18, 163-169]. Natural moisturizing factors (NMF) are found in the stratum corneum, which are a mixture of molecules with hygroscopic properties that favor water retention. Generally speaking, the skin's water content varies according to where the sample is taken; therefore, the content in the basal and suprabasal layers of living cells is approximately 75%, whilst the content in the stratum corneum is reduced by up to 10-15%. The relative humidity in the atmosphere, the epidermis' capacity to compensate the loss of water by evaporation and the intrinsic capacity of the stratum corneum to retain water are other factors which determine the skin's water content. Although the mechanisms which govern the transportation of water through the epidermis are still not completely clear, the existence of a continual exchange of water between the stratum corneum, the living cells in the underlying skin and the atmosphere seems to be clear, a process in which it is known that there are several factors involved. Of them, the composition of the stratum corneum, including its content of low molecular weight osmolytes or other molecules such as free amino acids, is particularly relevant, as the existence of a high concentration of Na+, K+and Cl− ions and a low concentration of water in the superficial part of the stratum corneum has been proven, which would generate gradients of water and solutes from the surface of the skin to the epidermal keratinocytes. The protein AQP-3 is considered the principal protein responsible for facilitating transepidermal permeability to protect the stratum corneum from drying due to the loss of water or from the dissipation of water gradients in the layer of epidermal keratinocytes.
AQP-3 is a member of the family of homologous integral membrane proteins and of the sub-class of aquaglyceroporins responsible for facilitating the transportation of water, glycerol, and other small solutes (e.g. urea), through biological membranes. In mammals, the family of aquaporins comprises 13 homologous proteins (AQP-1 to AQP-13) which can be classified into 3 groups: water channels, aquaglyceroporins and unorthodox aquaporins. Water channels can only transport water, aquaglyceroporins can transport water and glycerol, and, on occasions other small solutes; the aquaporins in the third group have specific properties, or they have not been clarified [Rojek A. et al., “A current view of the mammalian aquaglyceroporins” Annu. Rev. Physiol., (2008), 70, 301-327]. A wide spectrum of aquaporins can be found in mammals' skin, AQP-3 being the most abundant in human epidermis [Sougrat R. et al., “Functional expression of AQP-3 in human skin epidermis and reconstructed epidermis”, J. Invest. Dermatol., (2002), 118, 678-685]. AQP-3 is present not only in skin but also in tissue in the urinary tract, in the respiratory tract, in the digestive tract and in others, such as in collecting ducts.
There are different hypotheses of the molecular mechanisms through which AQP-3 acts. It is believed that the transportation of water in the skin occurs along an osmotic gradient under the stratum corneum, where permeability is mediated by AQP-3. The variations in the pH in the different layers of the skin, with values which can vary from 5 to 7 under the stratum corneum, allow the permeability of the skin to be modulated, as is the case of the marked impermeability in the granulo-corneoepidermal interface. In this context, the transported water would have an immobilizing effect on the layers of viable epidermal cells, which would promote hydration of the cutaneous layers which lay beneath the stratum corneum. There is a low concentration of water and a high concentration of solutes in the latter, responsible for generating a gradient of water and solutes between the outermost layer of the skin and the layer of viable keratinocytes [Takenouchi M. et al., “Hydration characteristics of pathologic stratum corneum-evaluation of bound water”, J. Invest. Dermatol., (1986), 87, 574-576]. It is believed that AQP-3 participates in the improvement of transepidermal permeability to protect the stratum corneum from the evaporation of water from the surface of the skin. Another possibility is that AQP-3 has a role in the dispersion of water gradients through the width of the layer of epidermal keratinocytes. The discontinuity of the water content between the stratum granulosum and the stratum corneum enables the existence of highly organized lipid-water lamellar structures located between corneocytes, that are crucial structures for the maintenance of the skin's barrier of permeability.
One of the phenotypic features characteristic in knockout mice deficient in AQP-3 is the dryness of the skin, which is one of the best proofs of the roles of AQP-3 in the hydration of the stratum corneum and, therefore, of the epidermis. Other changes to the skin which accompany the deficiency of AQP-3 are a reduction in elasticity, a delay in the recovery of the barrier functions, and a delay in the healing time of wounds [Hara M. et al., “Glycerol replacement corrects defective skin hydration, elasticity, and barrier function in aquaporin-3-deficient mice”. Proc. Natl. Acad. Sci. USA., (2003), 100, 7360-7365; Ma T. et al., “Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3”. J. Biol. Chem., (2002), 277, 17147-17153; Hara M. et al., “Selectively reduced glycerol in skin of aquaporin-3-deficient mice may account for impaired skin hydration, elasticity, and barrier recovery”. J. Biol. Chem., (2002), 277, 46616-46621]. AQP-3 also has an important role in the regulation of the differentiation and proliferation of keratinocytes [Bellemère G. et al., “Retinoic acid increases aquaporin 3 expression in normal human skin”. J. Invest. Dermatol., (2008), 128, 542-548], contributing to the maintenance of the skin barrier function. AQP-3 co-localizes with phospholipase D2 in caveolin-rich membrane microdomains, and provides phospholipase 2 with glycerol to generate phosphatidylglycerol, which, in turn, can initiate early differentiation [Zheng X. et al., “Aquaporin 3 colocates with phospholipase d2 in caveolin-rich membrane microdomains and is downregulated upon keratinocyte differentiation”. J. Invest. Dermatol., (2003), 121, 1487-1495].
One of the causes of the different conditions, disorders and diseases of the skin and/or mucous membranes is a reduction of the skin's water content. Among them we can highlight atopic dermatitis [Watanabe M. et al., “Functional analyses of the superficial stratum corneum in atopic xerosis”. Arch. Dermatol., (1991), 127, 1689-1692], eczema [Thune P., “Evaluation of the hydration and the water-holding capacity in atopic skin and so-called dry skin”. Acta Derm. Venereol. Suppl. (Stockh)., (1989), 144, 133-135], psoriasis [Tagami H., “Quantitative measurements of water concentration of the stratum corneum in vivo by high-frequency current”. Acta Derm. Venereol. Suppl. (Stockh), (1994), 185, 29-33], plantar hyperkeratosis, senile xerosis, [Horii I. et al., “Stratum corneum hydration and amino acid content in xerotic skin”. Br. J. Dermatol., (1989), 121, 587-592], hereditary ichthyosis [Hara M. et al., “Amelanotic acral melanoma masquerading as fibrous histiocytic tumours. Three case reports”. Acta Derm. Venereol., (1993), 73, 283-285], changes to the epidermis such as spongiosis [Boury-Jamot M. et al., “Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?”. Biochim. Biophys. Acta, (2006), 1758, 1034-1042] or vaginal dryness [KR20080024868 A] among others. All these conditions, disorders and diseases are, therefore, treatable with compounds which modulate AQP-3.
A relationship between AQP-3 and the skin's aging and photoaging has also been established. It has been proven that the epidermis experiences a reduction in AQP-3 expression in accordance with age and exposure to solar radiation [Dumas M. et al., “Histological variation of Japanese skin with ageing”. Int. J. Cosm. Sci., (2005), 27, 47-50].
Therefore, collagen fibers and hydration in the skin and/or mucous membranes are of great importance in maintaining the skin's balance and being able to reduce, delay and/or prevent the signs of aging and/or photoaging. It is important to have products available, whose effects are intended to maintain the levels of collagen and/or the hydration of the skin, and the maintenance of a smooth and firm appearance of the skin reducing, delaying and/or preventing the signs of aging and/or photoaging. The maintenance of a high collagen content in the skin or hydration of the skin can be achieved in many different ways. On the one hand, substances which induce collagen synthesis to counteract the negative effects of the skin's degradation with age can be used. Substances which modulate AQP-3 to increase the skin's hydration can also be used.
In the prior art there are active ingredients effective as instigators of collagen synthesis, such as ascorbic acid and its derivatives, in particular, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, and ascorbyl alpha- and beta-glucoside, retinol and derivatives of retinol such as retinoic acid, retinal, retinol, retinyl acetate, retinyl palmitate or plant extracts such as extracts of Aloe spp or Centella spp. The group of active ingredients frequently used to induce collagen synthesis also includes peptides and peptide derivatives such as carnitine, carnisine, peptides including peptides derived from matrikine (e.g. lysyl-threonyl-threonyl-lysyl-serine). In addition, compounds such as asiatic acid, madecassic acid, madecassoside, asiaticoside, extracts of Centella asiatica, niacinamide, astaxanthin, glucans e.g. from yeast and oat (Avena sativa), extract of soy (Glycine max), soy isoflavones e.g. genistein, daidzein, rutin, chrysin, morin, alkaloids of the areca nut, forskolin, betulinic acid, extracts of Plantago spp, TGF-beta, extracts of Ginkgo biloba, glutamine, and glycolic acid are used as stimulators of collagen synthesis.
There are also a number of compounds on the market capable of increasing the levels of aquaporins in the skin to palliate the symptoms of disorders related to its deficiency. The cosmetic and pharmaceutical industry is aware of this and has made considerable efforts to find molecules or extracts which bring about an increase in AQP-3 expression in the skin, such as xanthine, caffeine, ginsenosides, vitamin B3 or niacin [US 2007/0009474 A1], vitamin A or retinoic acid/all-trans retinoic acid (ATRA) [Bellemère G. et al., “Retinoic acid increases aquaporin 3 expression in normal human skin”. J. Invest. Dermatol., (2008), 128, 542-548], tocopheryl retinoate [JP 2006-290873 A], steroid derivatives, specifically the use of ecdysteroids [U.S. Pat. No. 5,609,873 A; U.S. Pat. No. 7,060,693 B1], glyceryl glucosides [WO 2007/124991 A1], glyceryl glycosides [US 2009/0130223 A1], peptides derived from the sequence of aquaporins [FR 2925500 A1], certain synthetic peptides [FR 2925501 A1], extract of Ajuga turkestanica [EP 1231893 B1], extract of Pyrus malus [FR 28899949 A1], extract of Vanda coerulea [FR 2928090 A1], extracts of brown algae such as Undaria pinnatifida [FR 2903015 A1], extract of Laminaria digitata [EP 1994923 A2], extract of Piptadenia colubrina[WO 2009/106934 A1], extracts of plants from the Tropaeolaceae family and of the species Crocus sativus [JP 2004-168732 A; JP 2005-343882 A] or extract of Punica granatum [FR 2831058 A1], among others.
However, despite the arsenal of existing compounds and/or extracts, the cosmetic, pharmaceutical and food sector is still interested in developing alternatives to the compounds known in the prior art, capable of stimulating collagen synthesis and/or increasing the hydration of the skin and/or mucous membranes.